U.S. patent application number 15/481323 was filed with the patent office on 2017-11-16 for methods for in vivo identification of endogenous mrna targets of micrornas.
The applicant listed for this patent is Duke University. Invention is credited to Jack D. Keene, Patrick J. Lager.
Application Number | 20170327866 15/481323 |
Document ID | / |
Family ID | 39107454 |
Filed Date | 2017-11-16 |
United States Patent
Application |
20170327866 |
Kind Code |
A1 |
Keene; Jack D. ; et
al. |
November 16, 2017 |
METHODS FOR IN VIVO IDENTIFICATION OF ENDOGENOUS MRNA TARGETS OF
MICRORNAS
Abstract
A method of generating a gene expression profile of noncoding
regulatory RNA (ncRNA; e.g. a microRNA) in a cell in vivo, is
carried out by: (a) partitioning from a cell at least one
mRNA-protein (RNP) complex, the RNP complex comprising: (i) an RNA
binding protein (RNABP) or RNA associated protein, (ii) at least
one mRNA bound to or associated with said protein, and (iii) at
least one ncRNA bound to or associated with said protein, and then
(b) identifying at least one ncRNA in at least one RNP complex,
thereby to produce a gene expression profile comprising the
identity of an ncRNA in an RNP complex.
Inventors: |
Keene; Jack D.; (Durham,
NC) ; Lager; Patrick J.; (Efland, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Family ID: |
39107454 |
Appl. No.: |
15/481323 |
Filed: |
April 6, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12438383 |
Jun 24, 2010 |
9617581 |
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PCT/US2007/018793 |
Aug 24, 2007 |
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15481323 |
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60823581 |
Aug 25, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6809 20130101;
C12Q 1/6809 20130101; C12Q 1/6804 20130101; C12Q 1/6804 20130101;
C12Q 2525/207 20130101; C12Q 2522/101 20130101; C12Q 2522/101
20130101; C12Q 2525/207 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/68 20060101 C12Q001/68 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under grant
number CA79907 from the National Institutes of Health. The U.S.
Government has certain rights in the invention.
Claims
1. A method of generating a gene expression profile of noncoding
regulatory RNA (ncRNA) in a cell in vivo, comprising the steps of:
(a) partitioning from a cell at least one mRNA-protein (RNP)
complex, said RNP complex comprising: (i) an RNA binding protein
(RNABP) or RNA associated protein, (ii) at least one mRNA bound to
or associated with said protein, and (iii) at least one ncRNA bound
to or associated with said protein, and then (b) identifying at
least one ncRNA in at least one RNP complex, thereby to produce a
gene expression profile comprising the identity of an ncRNA in an
RNP complex.
2. The method of claim 1, wherein said ncRNA is a microRNA.
3. The method of claim 1, said step of partitioning comprising:
contacting a biological sample comprising said RNP complex from the
cell with at least one ligand that specifically binds at least one
component of the RNP complex; separating the RNP complex by binding
the ligand with an antibody specific for the ligand, wherein the
antibody is attached to a solid support; and collecting the RNP
complex by removing the RNP complex from the solid support.
4. The method of claim 1, wherein said mRNA in said RNP complex is
predetermined.
5. The method of claim 1, further comprising the step of: (c)
identifying the mRNA in the RNP complex, thereby to produce a gene
expression profile further comprising the identity of the mRNA
associated with said miRNA.
6. The method of claim 1, wherein said mRNA encodes a protein
selected from the group consisting of amyloid protein, amyloid
precursor protein, angiostatin, endostatin, METH-1, METH-2, Factor
IX, Factor VIII, collagen, cyclin dependent kinase, cyclin D1,
cyclin E, WAF1, cdk4 inhibitor, MTS1, cystic fibrosis transmembrane
conductance regulator gene, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,
IL-17, erythropoietin, G-CSF, GM-CSF, M-CSF, SCF, thrombopoietin,
BNDF, BMP, GGRP, EGF, FGF, GDNF, GGF, HGF, IGF-1, IGF-2, KGF,
myotrophin, NGF, OSM, PDGF, somatotrophin, TGF-beta, TGF-alpha,
VEGF, interferon, TNF-alpha, TNF-beta, cathepsin K, cytochrome
p-450, famesyl transferase, glutathione-s transferase, heparanase,
HMG CoA synthetase, n-acetyltransferase, phenylalanine hydroxylase,
phosphodiesterase, ras carboxyl-terminal protease, telomerase, TNF
converting enzyme, E-cadherin, N-cadherin, selectin, CD40, 5-alpha
reductase, atrial natriuretic factor, calcitonin, corticotrophin
releasing factor, glucagon, gonadotropin, gonadotropin releasing
hormone, growth hormone, growth hormone releasing factor,
somatotropin, insulin, leptin, luteinizing hormone, luteinizing
hormone releasing hormone, parathyroid hormone, thyroid hormone,
thyroid stimulating hormone, antibodies, CTLA4, hemagglutinin, MHC
proteins, VLA-4, kallikrein-kininogen-kinin system, CD4, sis, hst,
ras, abl, mos, myc, fos, jun, H-ras, ki-ras, c-fms, bcl-2, L-myc,
c-myc, gip, gsp, HER-2, bombesin receptor, estrogen receptor, GABA
receptor, EGFR, PDGFR, FGFR, NGFR, GTP-binding regulatory proteins,
interleukin receptors, ion channel receptors, leukotriene receptor
antagonists, lipoprotein receptors, opioid pain receptors,
substance P receptors, retinoic acid and retinoid receptors,
steroid receptors, T-cell receptors, thyroid hormone receptors, TNF
receptors, tissue plasminogen activator; transmembrane receptors,
calcium pump, proton pump, Na/Ca exchanger, MRP 1, MRP2, P170, LRP,
cMOAT, transferrin, APC, brca1, brca2, DCC, MCC, MTS1, NF1, NF2,
nm23, p53 and Rb.
7. The method of claim 1, wherein said partitioning step comprises
partitioning a plurality of RNP complexes; and wherein said
identifying step comprises identifying a plurality of ncRNAs
associated with said plurality of RNP complexes; said method
further comprising: (c) identifying a plurality of mRNAs associated
with said plurality of RNP complexes; thereby to produce a gene
expression profile further comprising the identity of a subset of
ncRNAs associated with a subset of mRNAs.
8. The method of claim 1, wherein said cell is a plant cell.
9. The method of claim 1, wherein said cell is an animal cell.
10. The method of claim 1, wherein said cell is a bacterial
cell.
11. The method of claim 1, wherein said cell is a yeast cell.
12. The method of claim 1, wherein said cell is a protozoal
cell.
13. A method of identifying and/or confirming mRNA target(s) of one
or more microRNAs, the method comprising: (a) partitioning from a
biological sample at least one RNP complex, said complex containing
a subset of mRNAs associated with the RNP complex(es), and (b)
identifying a subset of microRNA associated with the RNP complex,
thereby determining the association between a microRNA and an mRNA
target.
14. The method of claim 13, wherein the step of partitioning
comprises capturing the RNP complex(es) on a solid support.
15. The method of claim 13, further comprising assaying activity of
at least one of the identified miRNA with respect to one or more of
the identified mRNAs.
16. The method of claim 13, further comprising predicting an mRNA
target of a microRNA in silico.
17. The method of claim 13, wherein the subset of mRNAs is
represented by less than 75% of all mRNAs in the biological
sample.
18. The method of claim 13, wherein the subset of mRNAs comprises
at least 2 mRNAs.
19. The method of claim 13, wherein the subset of miRNAs is
represented by less than 75% of all miRNAs in the biological
sample.
20. The method of claim 13, wherein the subset of miRNAs is
comprises at least 2 miRNAs.
21. The method of claim 13, wherein the subset of miRNAs and/or the
subset of the cellular miRNAs is/are identified by using a nucleic
acid array.
22. The method of claim 13, wherein the step of partitioning may
comprise contacting an mRNP complex with (i) an antibody that
specifically binds at least one component of the mRNP complex or
(ii) an ectopically expressed epitope-tagged RNA-binding protein or
an RNA-associated protein.
23. The method of claim 19, wherein the RNA-binding protein is a
native or tagged Hu protein or poly(A)-binding protein (PABP).
24. The method of claim 13, where the identified subset of the
microRNAs includes an miRNA selected from the group consisting of
miR-181a, miR-181b, miR-181c, miR-103, miR-107m miR-29c, miR-17-5p,
miR-106a, miR-19b, miR-16, let-7a, let-7c, let-7d, and let-7f.
25. A method of identifying and/or confirming mRNA target(s) of one
or more microRNAs, the method comprising: (a) obtaining a
biological sample comprising an mRNP complex; (b) contacting the
mRNP complex with (i) an antibody that specifically binds at least
one component of the mRNP complex or (ii) an ectopically expressed
epitope-tagged RNA-binding protein (RBP) or an RNA-associated
protein (RAP), (c) capturing the antibody, the RBP, or the RAP on a
solid support, thereby partitioning from the biological sample at
least one RNP complex, and (d) identifying a subset of microRNA
associated with the RNP complex(es), thereby determining the
association between a microRNA and an mRNA target.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of, and
claims priority to, U.S. application Ser. No. 12/438,383, filed
Jun. 24, 2010 (issued Apr. 11, 2017 as U.S. Pat. No. 9,617,581),
which is a 35 U.S.C. .sctn.371 national phase application of
International Application No. PCT/US2007/018793, filed Aug. 24,
2007, which claims the benefit, under 35 U.S.C. .sctn.119(e), of
U.S. Provisional Application No. 60/823,581, filed Aug. 25, 2006,
the entire contents of each of which are incorporated by reference
herein.
FIELD OF THE INVENTION
[0003] The present invention concerns methods of identifying
microRNAs and the corresponding mRNA targets thereof.
BACKGROUND OF THE INVENTION
[0004] MicroRNAs (miRNAs), together with RNA binding proteins
(RNABPs), constitute the primary regulators of eukaryotic
posttranscriptional gene expression and function in a broad range
of cellular processes. miRNAs are a large family of small noncoding
RNAs (ncRNAs) that repress gene expression by affecting the
stability or translation of target messenger RNAs (mRNAs) (1-3).
The current understanding of global miRNA targeting of mRNAs is
based upon computational predictions of complementary sequence
elements that are refined by considering evolutionary homologies
across multiple species (4). While these algorithms predict
hundreds of potential mRNA targets per miRNA, it is not certain
that each miRNA gains functional access to these target mRNAs in
the cell under a given set of conditions. Indeed, recent evidence
suggests that RNABPs can influence the regulatory fates of mRNAs
targeted by miRNAs in a condition-dependent manner (5, 6).
[0005] RNABPs, among the largest protein families encoded in
eukaryotic genomes, can regulate gene expression at multiple
posttranscriptional levels (7, 8). Like miRNAs, RNABPs also
function through binding specific RNA sequence motifs frequently
contained within untranslated regions (UTRs) of target mRNAs. When
occurring in the cytoplasmic compartment, these interactions may
determine mRNA localization, stability and/or translational
activation (9). It is becoming increasingly evident that the
posttranscriptional infrastructure is highly organized and utilizes
multiple cis-trans interactions to combinatorially regulate higher
order gene expression (7, 10). Global exploration of the in vivo
composition and organization of this posttranscriptional
infrastructure has only recently begun. A number of studies have
identified RNABPs associated with mRNA subsets that have similar
metabolic fates or encode functionally related proteins (7).
Several predicted functional interactions between miRNAs and mRNAs
have been confirmed using reporter systems, while a number of
primarily bioinformatics approaches have predicted the global
targeting of a substantial proportion of all cellular mRNAs by
miRNAs (2, 4, 11). While miRNAs are expected to act combinatorially
on their mRNA targets, the composition and organization of
endogenous miRNAs, mRNAs and RNABPs within messenger
ribonucleoprotein (mRNP) complexes are poorly understood.
SUMMARY OF THE INVENTION
[0006] A first aspect of the invention is a method of generating a
gene expression profile of noncoding regulatory RNA (ncRNA) in a
cell in vivo, comprising the steps of:
[0007] (a) partitioning from a cell at least one mRNA-protein (RNP
or mRNP) complex, said RNP complex comprising: (i) an RNA binding
protein (RNABP) or RNA associated protein, (ii) at least one mRNA
bound to or associated with said protein, and (iii) at least one
ncRNA bound to or associated with said protein, and then
[0008] (b) identifying at least one ncRNA in at least one mRNP
complex, thereby to produce a gene expression profile comprising
the identity of an ncRNA in an RNP complex.
[0009] In some embodiments the ncRNA is a microRNA.
[0010] In some embodiments, the invention provides a method of
identifying and/or confirming mRNA target(s) of one or more
microRNAs. Such a method comprises: [0011] (a) partitioning from a
biological sample at least one RNP complex, said complex containing
a subset of mRNAs associated with the RNP complex(es), and [0012]
(b) identifying a subset of microRNA associated with the RNP
complex(es), thereby determining the association between a microRNA
and an mRNA target. In some embodiment, the step of partitioning
comprising capturing the RNP complex(es) on a solid support. In
other embodiments, the method may further comprise the step of
assaying activity of at least one of the identified miRNA with
respect to one or more of the identified mRNAs. In further
embodiments, the method may comprise the step of predicting an mRNA
target of a microRNA using in silico methods (e.g., using the
TargetScanS algorithm) and validating the in silico results
experimentally as described above.
[0013] A subset of cellular mRNAs is a plurality of mRNAs that
includes less than, all mRNAs in the biological sample. In some
embodiments, such a subset is represented by less than 75%, 70%,
60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less of all
mRNAs. A subset of microRNAs is a plurality of microRNAs that
includes less than all microRNAs in the biological sample. In some
embodiments, such a subset is represented by less than 75%, 70%,
60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less of all
microRNAs. A subset of mRNAs comprises at least 2 but may comprise
3, 4, 5, 10, 15, 20 or more mRNAs. Likewise, a subset of microRNAs
comprises at least 2 but may comprise 3, 4, 5, 10, 15, 20 or more
microRNAs. The subsets of mRNAs and microRNA may be identified in
the methods of the invention, for example, by using a nucleic acid
array, e.g., a microarray (e.g., cDNA array).
[0014] In some embodiments, the step of partitioning may comprise
contacting an mRNP complex with (i) an antibody that specifically
binds at least one component of the mRNP complex or (ii) an
ectopically expressed epitope-tagged RNA-binding protein or an
RNA-associated protein. In some embodiments, the component of the
mRNP complex to which the antibody binds to an RNA-binding protein
or an RNA-associated protein present in the mRNP complex. In some
embodiments, such an RNA-binding protein is a native or tagged Hu
protein (e.g., HuR) or poly(A)-binding protein (PABP). In some
embodiments, the identified subset of the microRNAs includes an
miRNA selected from the group consisting of miR-181a, miR-181b,
miR-181c, miR-103, miR-107m miR-29c, miR-17-5p, miR-106a, miR-19b,
miR-16, let-7a, let-7c, let-7d, and let-7f.
[0015] In some embodiments the step of partitioning may comprise:
contacting a biological sample comprising said RNP complex from the
cell with at least one ligand that specifically binds at least one
component of the RNP complex; separating the RNP complex by binding
the ligand with an antibody specific for the ligand, wherein the
antibody is attached to a solid support; and collecting the RNP
complex by removing the RNP complex from the solid support.
[0016] In some embodiments the mRNA in said RNP complex is
predetermined; in some embodiments the method further comprises the
step of: (c) identifying the mRNA in the mRNP complex, thereby to
produce a gene expression profile further comprising the identity
of the mRNA associated with said miRNA.
[0017] Any suitable cell or cells can be used to carry out the
present invention, including but not limited to plant, animal,
bacterial, yeast, and protozoal cell.
[0018] In some embodiments the partitioning step comprises
partitioning a plurality of RNP complexes; the identifying step
comprises identifying a plurality of ncRNAs associated with the
plurality of RNP complexes; and the method further comprises: (c)
identifying a plurality of mRNAs associated with said plurality of
RNP complexes; thereby to produce a gene expression profile further
comprising the identity of a subset of ncRNAs associated with a
subset of mRNAs.
[0019] The present invention is explained in greater detail in the
drawings herein and the specification set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. HuR-associated mRNAs and miRNAs are discrete subsets
of total cellular RNA. Venn diagrams representing distinct (A)
messenger RNAs (mRNAs) and (B) microRNAs (miRNAs) present in total
cellular RNA, the PABP mRNP and the HuR mRNP. All RNA populations
were isolated as single samples from log phase Jurkat cells and
subsequently divided for analysis of mRNAs and miRNAs on specific
microarray platforms. Data was gathered from three biological
replicates and triplicate array analyses.
[0021] FIG. 2. Combinatorial posttranscriptional regulation
mediated by RNA binding proteins and miRNAs. Depiction of gene
expression networks localized to the nucleus (N) and cytoplasm. The
nuclear networks involve DNA binding transcription factors, while
the cytoplasmic networks involve RNABPs and miRNAs. In the nucleus,
multiple promoter elements can be regulated by transcription
factors. Posttranscriptional regulation primarily occurs through
interaction of RNA binding factors with 5' and 3' untranslated
regions (UTRs) of mRNAs. As shown, an mRNA subset regulated by a
given RNABP can be further subdivided into discrete mRNA
subpopulations that are also regulated in a combinatorial manner by
miRNAs. The coordinated outcome depicted here applies to functional
relationships among the encoded proteins or to the fates of the
associated mRNAs (stability/translational state).
[0022] The present invention is explained in greater detail in the
non-limiting specification and examples set forth below. The
disclosures of all United States patent references cited herein are
to be incorporated by reference herein in their entirety.
DETAILED DESCRIPTION
[0023] The present invention will now be described more fully with
reference to the accompanying drawings and specification, in which
preferred embodiments of the invention are shown. This invention
may, however, be embodied in different forms and should not be
construed as limited to the embodiments set forth herein. Rather,
these embodiments are provided so that this disclosure will be
thorough and complete, and will fully convey the scope of the
invention to those skilled in the art.
[0024] The terminology used in the description of the invention
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting of the invention. As used
in the description of the invention and the appended claims, the
singular forms "a", "an" and "the" are intended to include the
plural forms as well, unless the context clearly indicates
otherwise. Unless otherwise defined, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
[0025] "Messenger RNA" or "mRNA" as used herein has its ordinary
meaning in the art, and generally refers to an RNA transcribed from
DNA that carries encoded information to a site of protein synthesis
from that mRNA by translation. mRNA as used herein may be
unprocessed (pre-mRNA) or processed and hence the term is to
include both. mRNA as used herein may be from any suitable source,
typically vertebrate and preferably mammalian (e.g. human, dog,
cat, monkey, chimpanzee, mouse, rat, rabbit, etc.).
[0026] "Noncoding regulatory RNA" or "ncRNA" as used herein has its
ordinary meaning in the art. Examples include but are not limited
to piRNAs, microRNAs, ribosomal RNA (rRNA), small interfering RNA
(siRNA), small nuclear RNA (snRNA), small non-mRNA (snmRNA), small
nucleolar RNA (snoRNA), small temporal RNA (stRNA) and other RNAs
that interact with mRNAs to regulate the function thereof. See,
e.g., PCT Application Publication No. WO 2005/102298.
[0027] "MicroRNA" or "miRNA" as used herein has its ordinary
meaning in the art. Typically, a miRNA is a RNA molecule derived
from genomic loci processed from transcripts that can form local
RNA precursor miRNA structures. The mature miRNA usually has 20,
21, 22, 23, or 24 nucleotides, although in some cases, other
numbers of nucleotides may be present, for example, between 18 and
26 nucleotides. miRNAs are often detectable on Northern blots. The
miRNA has the potential to pair to flanking genomic sequences,
placing the mature miRNA within an imperfect RNA duplex which may
be needed for its processing from a longer precursor transcript. In
animals, this processing may occur through the action of Drosha and
Dicer endonucleases, which excise a miRNA duplex from the hairpin
portion of the longer primary transcript. The miRNA duplex
comprises the miRNA and a similar-sized segment, known as the
miRNA* (miRNA star), from the other arm of the stem-loop. The miRNA
is the strand that enters the silencing complex, whereas the miRNA*
degrades. In addition, miRNAs are typically derived from a segment
of the genome that is distinct from predicted protein-coding
regions. See, e.g., US Patent Application Publication No.
20060185027. miRNA as used herein may be from any suitable source,
typically vertebrate and preferably mammalian (e.g. human, dog,
cat, monkey, chimpanzee, mouse, rat, rabbit, etc.)
[0028] "mRNA binding protein" or "RNABP", along with RNA associated
proteins, as used herein have their ordinary meaning in the art,
and includes global RNABPs (those that bind to nearly all mRNAs
without distinguishing unique sequences), group-specific RNABPs
(those that associate with subsets of the global mRNA population),
and type-specific RNABPs (those that recognize a highly unique mRNA
sequence, in some cases present in only one mRNA, with high
specificity). See, e.g., J. Keene et al., Proc. Natl. Acad. Sci.
USA 98, 7018, 7021 (2001). Examples include but are not limited to
the ELAV/Hu family (e.g. HuR/Hu1,) eIF-4E, poly(A) binding
proteins, the PUMILIO family (e.g., Pum1), etc. Additional examples
are given in Keene et al., U.S. Pat. No. 6,635,422, at Table 1
therein, as follows:
TABLE-US-00001 TABLE 1 RNA Binding and RNA Associated Proteins SLBP
DAN TTP HeI-N1 Hel-N2 eIF-4A eIF-4B eIF-4G eIF-4E eIF-5 eIF-4EBP
MNK1 PABP p62 KOC p90 La Sm Ro U1-70K AUF-1 RNAse-L GAPDH GRSF
Ribosomal Po, P1, P2/L32 PM-Scl FMR Stauffen Crab 95 TIA-1 Upf1 RNA
BP1 RNA BP2 RNA BP3 CstF-50 NOVA-1 NOVA-2 CREBP GRBP SXL SC35 U2AF
ASF/SF2 ETR-1 IMP-1 IMP-2 IMP-3 ZBP LRBP-1 Barb PTB uPAmRNA BP
BARB1 BARB2 GIFASBP CYP mRNA BP IRE-BP p50 RHA FN mRNA BP AUF-1 GA
mRNA BP Vigillin ERBP CRD-BP HuA HuB HuC HuD hnRNP A hnRNP B hnRNP
C hnRNP D hnRNP E hnRNP F hnRNP G hnRNP H hnRNP K hnRNP L U2AF
[0029] RNABPs miRNAs, and mRNAs as used herein may be from any
suitable source, including bacteria, protozoa, plants and animals.
Plants may be vascular plants such as monocots and dicots, with
particular examples including but not limited to corn or maize,
wheat, soybean, canola, tomato, etc. Animals are typically
vertebrates and preferably mammals, with particular examples
including but not limited to human, dog, cat, monkey, chimpanzee,
mouse, rat, rabbit, etc. In particular embodiments of the invention
the RNABP, miRNA, and mRNA may all be from the same cell or tissue
from the same species of origin in native (non-transgenic)
form.
[0030] A "subset" of mRNA, ncRNA, miRNA has its ordinary meaning in
the art and is a plurality thereof, typically in an RNP complex. In
other words, subsets are defined by their ability to bind within or
to a particular RNP complex or subset of RNP complexes. The subset
will preferably be a quantitative or qualitative fraction of the
total population thereof of the cell. Furthermore, subsets within
subsets of mRNAs, ncRNAs, or miRNAs may be identified using the
invention. See, e.g., U.S. Pat. No. 6,635,422.
[0031] "RNA interference" or "RNAi" as used herein refers to
post-transcriptional process for attenuating gene expression in
which a natural (e.g., a miRNA) or artificial (e.g., an exogenously
administered double stranded RNA) interferes with the translation
of a target or corresponding mRNA, e.g., by hybridization to the
mRNA in a manner that interferes with normal translation
thereof.
[0032] The present invention can be carried out utilizing
techniques described in part in U.S. Pat. No. 6,635,422 to Keene et
al., or variations thereof that will be apparent to those skilled
in the art, with the provision where necessary of additional
selection and identification steps for ncRNAs such as miRNAs (which
can be carried out in essentially the same manner as the selection
and identification of mRNAs, typically with different probe sets or
microarray chips optimized for the selection and identification of
the ncRNAs such as miRNAs.
[0033] Except as otherwise indicated, standard methods may be used
for the production of cloned genes, expression cassettes, vectors,
and transformed cells and plants according to the present
invention. Such methods are known to those skilled in the art. See
e.g., J. Sambrook et al., Molecular Cloning: A Laboratory Manual
Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., 1989); F. M. Ausubel et al., Current Protocols In Molecular
Biology (Green Publishing Associates, Inc. and Wiley-Interscience,
New York, 1991).
[0034] Nucleotides and amino acids are represented herein in the
manner recommended by the IUPAC-IUB Biochemical Nomenclature
Commission, or (for amino acids) by three letter code, in
accordance with 37 C.F.R. .sctn.1.822 and established usage. See,
e.g., Patentin User Manual, 99-102 (November 1990) (U.S. Patent and
Trademark Office).
[0035] The terms "nucleic acid" or "nucleic acid sequence" may also
be used in reference to genes, cDNA, and mRNA encoded by a gene.
The term "gene" is used broadly to refer to any segment of DNA
associated with a biological function. Thus, genes include coding
sequences and/or the regulatory sequences required for their
expression. Genes also include non-expressed DNA segments that, for
example, form recognition sequences for other proteins. Genes can
be obtained from a variety of sources, including cloning from a
source of interest or synthesizing from known or predicted sequence
information.
[0036] As used herein, a nucleic acid molecule may be RNA (the term
"RNA" encompassing all ribonucleic acids, including but not limited
to ncRNA, pre-mRNA, mRNA, rRNA, hnRNA, snRNA and tRNA); DNA;
peptide nucleic acid (PNA, as described in, e.g., U.S. Pat. No.
5,539,082 to Nielsen et al., and U.S. Pat. No. 5,821,060 to
Arlinghaus et al.); and the analogs and modified forms thereof.
Nucleic acid molecules of the present invention may be linear or
circular, an entire gene or a fragment thereof, full-length or
fragmented/digested, "chimeric" in the sense of comprising more
than one kind of nucleic acid, and may be single-stranded or
double-stranded. Nucleic acid from any source may be used in the
present invention; that is, nucleic acids of the present invention
include but are not limited to genomic nucleic acid, synthetic
nucleic acid, nucleic acid obtained from a plasmid, cDNA,
recombinant nucleic acid, and nucleic acid that has been modified
by known chemical methods, as further described herein. Nucleic
acids may also be products of in vitro selection experiments (also
called aptamers) and other nucleic acid molecules useful for their
ability to bind or be bound by other ligands. See D. Kenan, TIBS
19, 57-64 (1994); L. Gold, et al., Annu. Rev. Biochem. 64, 763-798
(1995); S. E. Osborne and A. D. Ellington, Chem. Rev. 97, 349-370
(1997).
[0037] As summarized above, the present invention relates to in
vivo methods for partitioning RNP complexes from a cell. mRNP
complexes of the present invention is preferably from a biological
sample, such as a tissue sample, whole tissue, a whole organ (e.g.,
an entire brain, liver, kidney, etc.), bodily fluid sample, cell
culture, cell lysate, cell extract or the like. In a preferred
embodiment, the biological sample comprises or is obtained from a
population of cells. By a "population of cells" herein is meant at
least two cells, with at least about 10.sup.3 being preferred, at
least about 10.sup.6 being particularly preferred, and at least
about 10.sup.8 to 10.sup.9 being especially preferred. The
population or sample can contain a mixture of different cell types
from either primary or secondary cultures, or from a complex tissue
such as a tumor, or may alternatively contain only a single cell
type. In a preferred embodiment, cells that are proliferating are
used. Alternatively, non-proliferating cells may be used.
[0038] As summarized above, one aspect of the invention is an in
vivo method of partitioning endogenous cellular mRNA-binding
protein (mRNP) complexes. "Endogenous" is used herein to mean that
the mRNP complex forms in a cell (i.e., in vivo or in situ). The
mRNP complex may form in the cell naturally, i.e., the components
of the mRNP complex naturally occur in the cell and form the mRNP
complex. Alternatively, the mRNP complex forms in a cell, even
though one or more components of the complex is introduced into the
cell by, e.g., infection or transformation. For example, an mRNP
complex endogenously forms in a cell when a RNA-binding protein
that is a component of the mRNP complex is ectopically expressed in
the cell by (for example) transforming the cell or infecting the
cell with an expression vector that carries nucleic acid encoding
the protein, and a mRNP complex in which the protein binds is
formed.
[0039] The method, in one embodiment, comprises contacting a
biological sample that comprises at least one mRNP complex with a
ligand that specifically binds a component of the mRNP complex. The
component of the mRNP complex may be a RNA binding protein, a
RNA-associated protein, a nucleic acid associated with the mRNP
complex including the mRNA itself, ncRNAs, or another molecule or
compound (e.g., carbohydrate, lipid, vitamin, etc.) that associates
with the mRNP complex. A component "associates" with a mRNP complex
if it binds or otherwise attaches to the mRNP complex with a Kd of
about 10.sup.6 to about 10.sup.9. In a preferred embodiment, the
component associates with the complex with a Kd of about 10.sup.7
to about 10.sup.9. In a more preferred embodiment, the component
associates with the complex with a Kd of about 10.sup.8 to about
10.sup.9.
[0040] The ligand may be any molecule that specifically binds the
component of the mRNP complex. For example, the ligand may be an
antibody that specifically binds the component, a nucleic acid that
binds the component (e.g., an antisense molecule, a RNA molecule
that binds the component), or any other compound or molecule that
specifically binds the component of the complex. In certain
embodiments, the ligand may be obtained by using the serum of a
subject (i.e., a human or animal subject) that has a disorder known
to be associated with the production of mRNP-complex specific
antibodies or proteins. Examples of these disorders include
autoimmune disorders such as systemic lupus erythematosus ("lupus"
or SLE) and a number of cancers. In certain embodiments, the ligand
may be "tagged" with another compound or molecule in order to
facilitate the separation, observation or detection of the ligand.
In one embodiment of the invention, the ligand is "epitope tagged,"
as described in the art. Suitable tags are known in the art and
include but are not limited to biotin, the MS2 protein binding site
sequence, the U1snRNA 70 k binding site sequence, the U1snRNA A
binding site sequence, the g10 binding site sequence (commercially
available from Novagen, Inc., Madison, Wis., USA), and
FLAG-TAG.RTM. (Sigma Chemical, St. Louis, Mo., USA).
[0041] The mRNP complex may then be separated by binding the ligand
(now bound to the mRNP complex) to a binding molecule that
specifically binds the ligand. The binding molecule may bind the
ligand directly (i.e., may be an antibody or protein specific for
the ligand), or may bind the ligand indirectly (i.e., may be an
antibody or binding partner for a tag on the ligand). Suitable
binding molecules include but are not limited to protein A, protein
G, streptavidin. Binding molecules may also be obtained by using
the serum of a subject suffering from, for example, an autoimmune
disorder or cancer. In certain embodiments, the ligand is an
antibody that binds the component of the mRNP complex via the Fab
region of the antibody, and the binding molecule in turn binds the
Fc region of the antibody. The binding molecule will be attached to
a solid support, such as a bead, well, pin, plate or column, as
known in the art. Accordingly, the mRNP complex will be attached to
the solid support via the ligand and binding molecule.
[0042] The mRNP complex may then be collected by removing it from
the solid support (i.e., the complex is washed off the solid
support under appropriate stringency conditions, using suitable
solvents that may be determined by skilled artisans).
[0043] In certain embodiments of the invention, the mRNP complex
may be stabilized by cross-linking prior to binding the ligand
thereto. Cross-linking, as used herein, means covalently binding
(e.g., covalently binding the components of the mRNP complex
together). Cross-linking may be contrasted with ligand-target
binding, or binding molecule-ligand binding, which is generally
non-covalent binding. Cross-linking may be carried out by physical
means (e.g., by heat or ultraviolet radiation), or chemical means
(e.g., by contacting the complex with formaldehyde,
paraformaldehyde, or other known cross-linking agents), which means
are known or determinable by those skilled in the art. In other
embodiments, the ligand may be cross-linked to the mRNP complex
after binding the mRNP complex. In additional embodiments, the
binding molecule may be cross-linked to the ligand after binding to
the ligand. In yet other embodiments, the binding molecule may be
cross-linked to the solid support.
[0044] The skilled artisan will appreciate the present method
allows for the identification of a plurality of mRNP complexes
simultaneously (e.g., "en masse"). For example, a biological sample
may be contacted with a plurality of ligands specific for different
mRNP complex components. A plurality of mRNP complexes from the
sample will bind the various ligands. The plurality of mRNP
complexes can then be separated using appropriate binding
molecules, thus isolating the plurality of mRNP complexes. The mRNP
complexes and the mRNAs and ncRNAs contained within the complexes
may then be characterized and/or identified by methods described
herein and known in the art. Alternatively, the method may be
carried out on one sample numerous times, the inventive steps being
performed in a sequential fashion, with each iteration of steps
utilizing a different ligand.
[0045] As set forth above, a subset of mRNA and/or ncRNAs
identifies a pattern-recognition profile that is characteristic of
the RNA structural or functional networks in that sample. The
collection of mRNA and/or ncRNA subsets for any particular cell or
tissue sample constitutes a gene expression profile, and more
specifically a ribonomic gene expression profile, for that cell or
tissue. It will be appreciated that ribonomic expression profiles
may differ from cell to cell, depending on the type of cell in the
sample (e.g., what species or tissue type the cell is), the
differentiation status of the cell, the viability of the cell
(i.e., if the cell is infected or if it is expressing a deleterious
gene, such as an oncogene, or if the cell is lacking a particular
gene or not expressing a particular gene), the specific ligands
used to isolate the mRNP complexes, etc. Thus, the ribonomic
expression profile of a cell may be used as an identifier for the
cell, enabling the artisan to compare and distinguish profiles or
subprofiles of different cells. The genes identified by the RNAs
present in each ribonomic pattern form distinct subsets that may be
associated with a particular cell cycle, stage of differentiation,
apoptosis or stress induction, viral infection, or cancer.
[0046] cDNAs may be used to identify mRNP complexes partitioned
with a ligand or ligands specific for a component of the mRNP
complex. cDNA microarray grids, for example, may be used to
identify mRNA and ncRNA subsets en masse. Alternatively, genomic
microarrays (e.g., microarrays wherein the target nucleic acids may
contain introns and exons) may be used. Therefore, each gene or
target nucleic acid being examined on a microarray has a precise
address that can be located, and the binding can be quantitated.
Microarrays in the form of siliconized chips or those based upon
cDNA blots on nylon or nitrocellulose are commercially available.
Glass slides can also be customized with oligonucleotides or DNAs
for detection of complementary RNA sequences. In all of these
cases, the hybridization platforms allow identification of the
mRNAs and ncRNAs in a sample based upon the stringency of binding
and washing. This has been referred to as "sequencing by
hybridization." Although microarray technology is one method of
analysis, it is only one way to identify and/or sequence the mRNAs
and ncRNAs in the mRNA and ncRNA subset. Alternative approaches
include but are not limited to differential display, phage
display/analysis, SAGE or simply preparing cDNA libraries from the
mRNA and ncRNA preparation and sequencing all members of the
library.
[0047] Methods for DNA sequencing which are well known and
generally available in the art may be used to practice any of the
embodiments of the invention. The methods may employ such enzymes
as the Klenow fragment of DNA polymerase I, SEQUENASE.RTM. (US
Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of polymerases and proofreading exonucleases such as
those found in the ELONGASE Amplification System marketed by
Gibco/BRL (Gaithersburg, Md.). Preferably, the process is automated
with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,
Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,
Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin
Elmer).
[0048] In a preferred embodiment, amplification of the mRNA and
ncRNA isolated according to the present invention, and/or the cDNA
obtained from the mRNA is not carried out during the identification
of the nucleic acid, and is not necessary or required by the
present invention. However, the skilled artisan may choose to
amplify the nucleic acid that is the subject of identification
(e.g., the nucleic acid being identified via microarray analysis
and/or sequencing) for convenience, as a matter of preference,
and/or to comply with the specification/instructions of certain
commercially available microarrays or microarray analysis systems.
Thus, if desired, the nucleic acid may be amplified according to
any of the numerous known nucleic acid amplification methods that
are well-known in the art (e.g., PCR, RT-PCR, QC-PCR, SDA, and the
like).
[0049] Methods of the present invention may be carried out in
several ways, according to the needs of the practitioner and the
purpose for which the invention is carried out. For example, in one
embodiment, mRNA-binding protein complexes that are unique to a
cell type of interest are identified. In an example of such an
embodiment, an antibody that is specific for the mRNP complex can
be used to immunoprecipitate the complex with its associated mRNAs
and ncRNAs. The RNAs may then identified to form the ribonomic
expression profile of that cell type, or alternatively may be
isolated for (as an example) drug screening. The mRNA and/or ncRNA
candidates for post-transcriptional regulation may be analyzed en
masse, as a subset, for changes in mRNA and/or ncRNA stability
during the cell cycle or developmental events. In certain
embodiments, the methods may be carried out by isolating nuclei
from cells undergoing developmental or cell cycle changes,
performing nuclear run-off assays according to known techniques to
obtain transcribing mRNAs and/or ncRNAs, and then comparing the
transcribing mRNAs and/or ncRNAs with the global mRNA and/or ncRNA
levels in the same cells using cDNA microarrays. These methods thus
provide the ability to distinguish transcriptional from
post-transcriptional effects on steady state mRNA and/or ncRNA
levels en masse.
[0050] In another embodiment, cells in culture are transformed to
express a RNA-binding protein (RBP) or RNA-associated protein (RAP)
that will associate with particular mRNAs and ncRNAs only in a cell
type of interest. DNA encoding the RBP or RAP may be carried by a
recombinant vector (e.g., a plasmid, a viral vector) and
transformed into the cell by known means, after which the RBP or
RAP is expressed in the cell. Any RBP or RAP can be used, as
described further herein. The protein may be in its native form, or
it may be tagged (e.g., epitope tagged) for easy recovery from the
cell. Detection of multiple RNA targets in vivo that are bound or
associated with RBPs or RAPs may be carried out by using accessible
epitopes, if necessary, but preferably is carried out without tags.
In cases where the epitopes on the RBPs or RAPs are inaccessible or
obscured, epitope tags on ectopically expressed recombinant
proteins may be used. The transformed cell may be mixed with other
cell types or may be implanted in an animal or human subject. A
ligand (e.g., an antibody) that is specific for the protein can
used to immunoprecipitate the protein with its associated messenger
RNAs from an extract of a tissue containing the transformed cell.
The mRNA and ncRNA complexes and its associated RNAs may then
identified to form the expression profile of that cell type or is
otherwise analyzed (e.g., for drug development).
[0051] In still another embodiment, a specific cell type in an
animal is engineered with one or more cell-type specific gene
promoters to express a RBP or RAP in the cell type of interest. As
set forth above, the gene promoter and the RBP or RAP may be
carried on one or more vectors and transformed into the cell, where
the RBP or RAP is expressed. In one embodiment, a ligand (e.g., an
antibody) that is specific for this protein can used to
immunoprecipitate the protein with its attached or associated mRNAs
and ncRNAs from an extract of a tissue containing the cell type of
interest. The RNAs are then identified to form the expression
profile of that cell type or isolated, e.g., for drug
development.
[0052] RNA binding proteins (RBPs) and RNA-associated proteins
(RAPs) useful in the practice of the present invention are known in
the art, or may alternatively be identified and discovered by
methods described herein. RNA binding proteins are now known to be
involved in the control of a variety of cellular regulatory and
developmental processes, such as RNA processing and
compartmentalization, RNA stabilization, mRNA translation and viral
gene expression. RNA binding proteins include poly A-binding
protein ("PABP," which gives rise to a subset of the total mRNA
population that is quantitatively different from the total mRNA
population), and other general RNA binding proteins, as well as
RNA-binding proteins that are attached to only one or a few
messenger RNAs in a particular cell type. Other useful proteins are
autoantibodies reactive with RNA and RNA-binding proteins.
[0053] Examples of useful RNA binding proteins and RNA associated
proteins are described above and include the four ELAV/Hu mammalian
homologues of the Drosophila ELAV RNA-binding protein (Good (1995)
Proc. Natl. Acad. Sci. USA 92, 4557-4561; Antic and Keene, supra.
HuA (HuR) is ubiquitously expressed while HuB, HuC and HuD (and
their respective alternatively-spliced isoforms) are predominantly
found in neuronal tissue, but can also be expressed as tumor
cell-specific antigens in some small cell carcinomas,
neuroblastomas, and medulloblastomas (reviewed in Keene (1999)
Proc. Natl. Acad. Sci. USA 96, 5-7). All Hu proteins contain three
RNA-recognition motifs (RRMs), which confer their binding
specificity for AREs (Antic and Keene, supra; Kenan et al. (1991)
Trends Biochem. Sci. 16, 214-220; Burd and Dreyfuss (1994) Science
265, 615-621). The evidence for ARE binding by Hu proteins began
with the identification of an AU-rich binding consensus sequence
from a randomized combinatorial RNA library that was screened with
recombinant HuB (Levine et al. (1993) Mol. Cell Biol. 13,
3494-3504; Gao et al. (1994) Proc. Natl. Acad. Sci. USA 91,
11207-11211). These and other studies demonstrated that Hu proteins
bind in vitro to several ARE-containing ERG mRNAs including c-myc,
c-fos, GM-CSF and GAP-43 (Levine et al. (1993) Mol. Cell Biol. 13,
3494-3504; Gao et al. (1994) Proc. Natl. Acad. Sci. USA 91,
11207-11211; King et al. (1994) J. Neurosci. 14, 1943-1952; Liu et
a. (1995) Neurology 45, 544-550; Ma et al (1996) J. Biol. Chem.
271, 8144-8151; Abe et al. (1996) Nucleic Acids Res. 24, 2011-2016;
Chung et al. (1997) J. Biol. Chem. 272, 6593-6598; Fan and Steitz
(1998) EMBO J. 17, 3448-3460; Antic et al. (1999) Genes Dev. 13,
449-461).
[0054] The binding of Hu proteins to ARE-containing mRNAs can
result in the stabilization and increased translatability of the
mRNA transcripts (Jain et al. (1997) Mol. Cell Biol. 17, 954-962;
Levy et al. (1998) J. Biol. Chem. 273, 6417-6423; Fan and Steitz
(1998) EMBO J. 17, 3448-3460; Peng et al. (1998) EMBO J. 17,
3461-3470). The neuron-specific Hu proteins are one of the earliest
neuronal markers produced in teratocarcinoma cells following
retinoic acid (RA)-treatment to induce neuronal differentiation
(Antic et al., supra; Gao and Keene (1996) J. Cell Sci. 109,
579-589).
[0055] In one embodiment, the ligand used to carry out the
invention is a RNA binding protein selected from the RNA
Recognition Motif (RRM) family of cellular proteins involved in
pre-messenger RNA processing. One example of such a protein is the
U1A snRNP protein. More than 200 members of the RRM superfamily
have been reported to date, the majority of which are ubiquitously
expressed and conserved in phylogeny (Query et al, Cell (1989) 57:
89-101; Kenan et al, Trends Biochem. Sci. (1991) 16: 214-220). Most
are known to have binding specificity for polyadenylate mRNA or
small nuclear ribonucleic acids (e.g. U1, U2, etc.) transfer RNAs,
5S or 7S RNAs. They include but are not limited to hnRNP proteins
(A, B, C, D, E, F, G, H, I, K, L), RRM proteins CArG, DT-7, PTB,
K1, K2, K3, HuD, HUC, rbp9, elF4B, sxl, tra-2, AUBF, AUF, 32KD
protein, ASF/SF2, U2AF, SC35, and other hnRNP proteins.
Tissue-specific members of the RRM family are less common,
including IMP, Bruno, AZP-RRMI, X16 which is expressed in pre-B
cells, Bj6 which is a puff-specific Drosophila protein and ELAV/Hu,
which are neuron specific.
[0056] RNA-binding and RNA-associated proteins useful in the
practice of the present invention include but are not limited to
those described above.
[0057] Antibodies that specifically bind mRNP complexes are known
and described in, for example, U.S. Pat. No. 6,635,422 to Keene et
al.
[0058] The present invention can be used to identify ncRNAs such as
miRNAs that bind to, or interact with in an RNP, an mRNA encoding
for a protein with which progression of a disease is associated
(protein of interest). Such an mRNA may be predetermined, or
identified from a subpopulation, subset of mRNAs generated by the
methods of the present invention (e.g., where a cell known to
express the protein of interest is utilized in carrying out the
method). Examples of mRNAs encoding such proteins include but are
not limited to those described in U.S. Pat. No. 6,503,713 to Rana
at section 5.1 therein. Specific examples include but are not
limited to mRNAs that encode proteins such as amyloid protein,
amyloid precursor protein, angiostatin, endostatin, METH-1, METH-2,
Factor IX, Factor VIII, collagen, cyclin dependent kinase, cyclin
D1, cyclin E, WAF1, cdk4 inhibitor, MTS1, cystic fibrosis
transmembrane conductance regulator gene, IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
IL-15, IL-16, IL-17, erythropoietin, G-CSF, GM-CSF, M-CSF, SCF,
thrombopoietin, BNDF, BMP, GGRP, EGF, FGF, GDNF, GGF, HGF, IGF-1,
IGF-2, KGF, myotrophin, NGF, OSM, PDGF, somatotrophin, TGF-.beta.,
TGF-alpha, VEGF, interferon, INF-alpha, TNF-beta, cathepsin K,
cytochrome p-450, famesyl transferase, glutathione-s transferase,
heparanase, HMG CoA synthetase, n-acetyltransferase, phenylalanine
hydroxylase, phosphodiesterase, ras carboxyl-terminal protease,
telomerase, TNF converting enzyme, E-cadherin, N-cadherin,
selectin, CD40, 5-alpha reductase, atrial natriuretic factor,
calcitonin, corticotrophin releasing factor, glucagon,
gonadotropin, gonadotropin releasing hormone, growth hormone,
growth hormone releasing factor, somatotropin, insulin, leptin,
luteinizing hormone, luteinizing hormone releasing hormone,
parathyroid hormone, thyroid hormone, thyroid stimulating hormone,
antibodies, CTLA4, hemagglutinin, MHC proteins, VLA-4,
kallikrein-kininogen-kinin system, CD4, sis, hst, ras, abl, mos,
myc, fos, jun, H-ras, ki-ras, c-fms, bcl-2, L-myc, c-myc, gip, gsp,
HER-2, bombesin receptor, estrogen receptor, GABA receptor, EGFR,
PDGFR, FGFR, NGFR, GTP-binding regulatory proteins, interleukin
receptors, ion channel receptors, leukotriene receptor antagonists,
lipoprotein receptors, opioid pain receptors, substance P
receptors, retinoic acid and retinoid receptors, steroid receptors,
T-cell receptors, thyroid hormone receptors, TNF receptors, tissue
plasminogen activator; transmembrane receptors, calcium pump,
proton pump, Na/Ca exchanger, MRP 1, MRP2, P170, LRP, cMOAT,
transferrin, APC, brca1, brca2, DCC, MCC, MTS1, NF1, NF2, nm23, p53
and Rb. See, e.g., U.S. Pat. No. 6,503,713. Additional examples are
given in US Patent Application No. 2003/0073610, at Table 1
therein, and include but are not limited to mRNAs that encode
proteins involved in aberant protein deposition, such as:
alpha-synuclein (Parkinson's Disease); Amyloid-beta (Alzheimer's
Disease) Tau (Alzheimer's Disease), PrP (Prion Diseases);
huntingtin (Huntington's Disease); Ataxin-1 (Spinocerebellar
ataxia-1) Ataxin-2 (Spinocerebellar ataxia-2); Ataxin-3
(Spinocerebellar ataxia-3); Calcium channel (Spinocerebellar
ataxia-6); Ataxin-7 (Spinocerebellar ataxia-7); Androgen receptor
(Spinal and bulbar Muscular atrophy); Atrophin-1 (Dentatorubral
Pallidoluysian atrophy); SOD1 (Amyotropic lateral sclerosis);
Immunoglobulin light chain (Primary systemic amyloidosis);
Transthyretin (Famylial amyloid polyneuropathy; Senile systemic
amyloidosis); Serum amyloid A (Secondary systemic amyloidosis);
Islet amyloid polypeptide (Type 2 diabetes); Insulin
(Injection-localized amyloidosis); beta 2-microglobulin
(Hemodialysis-related amyloidosis); Cystatin-C(Hereditary cerebral
amyloid angiopathy); Gelsolin (Finnish hereditary systemic
amyloidosis); and Lysozyme.
[0059] ncRNAs and miRNAs identified by the methods of the present
invention are useful in native form or derivatized form as RNA
interference (RNAi) active agents, such as described in U.S. Pat.
No. 7,078,196; U.S. Pat. No. 6,503,713 (particularly section 5.2
therein); and US Patent Application 2004/0086884. Moreover, the
molecular interactions and interaction sites as defined using this
invention can provide validated targets for the development of
compounds and reagents such as interfering RNAs.
[0060] ncRNAs and miRNAs identified by the present invention are
useful in the production of proteins or peptides in vitro or in
vivo, where it is desired to downregulate the production of one or
more particular proteins or peptides (e.g., RNA interference during
a growth phase of bacterial, plant, animal, or yeast cells), and
then remove that downregulation during a subsequent production
phase. Such applications would be useful in treating diseases such
as those described herein and in the production of recombinant
proteins and peptides.
[0061] In some cases it is desired to inhibit or inactivate an
ncRNA or miRNA in vivo, for example where the downregulation of
expression of a protein is undesired or pathological. In such cases
ncRNAs and miRNAs of the invention are useful for designing
anti-miRNA or anti-ncRNA oligonucleotides (AMOs) that hybridize
thereto, which AMOs can be designed and synthesized in accordance
with known techniques in order to regulate the expression of a
given protein that is encoded by the mRNA target so defined. See,
e.g., J. Weiler et al., Anti-miRNA oligonucleotides (AMOs):
ammunition to target miRNAs implicated in human disease, Gene
Therapy 13: 496-502 (2006).
[0062] ncRNAS and miRNAs (or subsets and combinations thereof)
identified by the methods of the present invention, together with
the RNABPs and corresponding mRNAs to which they bind or with which
they are associated (or subsets thereof), are useful in providing
confirming data, validating data, or training data to refine models
and algorithms for identifying or generating hypothetical or
proposed miRNAs from known corresponding mRNAs, such as described
in US Patent Application 2006/0185027. For example, the data set
forth in Table 2 below illustrates the narrowing of a class of 1013
mRNAs predicted as potential miRNA targets by a commercial
algorithm to a smaller subset of 108 mRNAs, thereby speeding and
facilitating the identification of miRNA targets.
[0063] The present invention is explained in greater detail in the
following non-limiting Examples.
Example 1
A Discrete Subset of microRNAs and Predicted mRNA Targets are
Enriched Components of HuR mRNPs
[0064] To examine the RNA and protein components of endogenous
RNPs, we conducted a genome-wide analysis of miRNA and mRNA
populations associated with the regulatory RNA binding protein HuR
in the human Jurkat T cell line. The ubiquitously expressed
mammalian HuR protein is one of four members of the ELAV/Hu family
that all function primarily through association with AU-rich
elements (AREs) in the 3'UTRs of target mRNAs, resulting in
enhanced message stability and/or translation (12-14). Recently,
HuR has also been shown to derepress the microRNA miR-122
translational repression of cationic amino acid transporter 1
(CAT-1) mRNA in a human hepatocellular carcinoma cell line
subjected to stress conditions (6).
[0065] Here we report that discrete subsets of miRNAs and mRNAs are
associated with HuR in human Jurkat T cells and that the mRNA
subpopulation is highly enriched for computationally predicted
miRNA targets that encode many growth regulatory proteins. Among
the fourteen miRNAs found in the HuR mRNP are members of the
mir-17-92 cluster previously implicated as an oncogene, as well as
miR-16 which is reported to be associated with apoptosis, chronic
lymphocytic leukemia and ARE-mediated mRNA decay (5, 15-17). This
report is the first demonstration that subsets of miRNAs are
components of specific RNP complexes that are also enriched for
functionally related target mRNAs. As biologically derived
co-subsets, these HuR RNP-associated miRNAs and mRNAs provide a
greatly reduced sequence space in which to examine miRNA targeting
and the outcomes of predicted miRNA:mRNA interactions in a given
cellular context. We address possible combinatorial relationships
between RNABPs and miRNAs, regulation of the targeted mRNA
subpopulations and resultant gene expression networks. We propose
that mRNAs regulated by one posttranscriptional mechanism such as
RNABPs may have preferentially evolved or acquired additional
posttranscriptional regulators to diversify and coordinate the
outcomes of gene expression.
[0066] In this study, endogenous HuR and poly(A)-binding protein
(PABP) mRNPs were directly isolated from Jurkat cell lysates by
immunoprecipitation with specific antibodies. RNA extracted from
these mRNPs, as well as from total cellular RNA, was analyzed on a
commercially available mRNA microarray platform and a previously
validated array platform specific for miRNAs (see Materials and
Methods). The results demonstrate that HuR associates in vivo with
a distinct subset of both the total cellular mRNAs and miRNAs (FIG.
1). Interestingly, HuR associates with a larger fraction of
cellular miRNAs (23%) than mRNAs (10%), while PABP associates
proportionally with each (85% and 84%, respectively). Two other
RNABPs were also tested and did not produce these RNA subsets (data
not shown). The 14 miRNAs associated with HuR are grouped into 7
miRNA families out of the approximately 62 known families based on
seed sequence conservation (Table 1A) (18, 19). Several of the
HuR-associated miRNAs have previously been reported to function in
processes also associated with ARE-mediated RNA stability and
translation in which Hu proteins are well established regulators.
These include effects on cellular proliferation and apoptosis by
miR-16, the miR-17 oncomir cluster (including miR17-5p and miR-19b)
and the let-7 family (13, 15, 16, 20, 21). In addition, miR-16 has
been implicated in TNF-.alpha. mRNA instability mediated through an
ARE sequence motif that is also an expected binding site of the
RNABPs tristetraprolin (TTP) and HuR (5).
[0067] To address the targeting of mRNAs by the HuR-associated
miRNA subset, we utilized the TargetScanS algorithm that relies
upon evolutionary conservation of miRNA seed matches to predict
target mRNAs (19). The 7 HuR-associated miRNA families are
predicted to target 1084 mapped mRNAs conserved in the 3'UTRs of 5
vertebrate species (see Materials and Methods). 439 of these mRNAs
are expressed in Jurkat cell total RNA, while 108 are associated
with HuR (Table 2). The association of these 108 mRNAs with HuR
represents an exceptional enrichment of miRNA targets as determined
using TargetScanS and was confirmed by a hypergeometric statistical
test that yields a P value of 2.6e-16 (probability that miRNA
target enrichment in the HuR mRNP occurs by chance). Additional
analyses of groups of 7 miRNA family sets randomly chosen from all
miRNAs (representing 62 families) also show target enrichment to
mRNAs in the HuR mRNP. Interestingly, only an additional 130 mRNAs
are added to the targeted subpopulation in this case (data not
shown). Taken together, these data demonstrate that the discrete
subpopulation of HuR-associated mRNAs is preferentially targeted by
miRNAs. This is consistent with the fact that many mRNA targets of
Hu family proteins encode early response gene proteins involved in
cell growth and differentiation, processes also implicated in miRNA
regulation (1, 2, 12, 13, 22).
[0068] Gene ontology analysis of mRNAs predicted to be targets of
HuR-associated miRNAs reveals an enrichment of several functional
annotation groups (Table 3). HuR-associated miRNA targets encode
proteins that show statistically significant enrichment in 10
annotation groups, while those expressed globally in Jurkat total
RNA are enriched in 16 categories. Interestingly, only 3 functional
groups overlap between the two analyses, suggesting again that the
association of predicted miRNA targets with HuR is not random and
represents enrichment in distinct functional classes. The predicted
mRNA targets of miRNAs found to be associated with HuR are
predominantly enriched in functional categories relating to
transcriptional regulation and RNA metabolism, two areas also
attributed to HuR regulation (13, 23). These findings are
consistent with an interconnection and potential coordination of
transcriptional and posttranscriptional regulatory networks by
RNABPs and miRNAs (7, 22-24).
[0069] Current understanding of the global populations of mRNAs
that may be directly targeted by miRNAs relies almost entirely upon
computational approaches, and these algorithms have significantly
advanced functional predictions of these interactions. However,
reliance upon strict evolutionary conservation in these predictions
may overlook mRNA targets that are species specific. Isolation of
endogenously associated miRNA:mRNA subpopulations as reported here
substantially reduces the sequence space to be examined for
productive interactions, many of which may depend upon cell type,
growth condition or intracellular context. As an analogy, RNABPs,
and RNPs in general, have been demonstrated to exhibit condition
dependent association with mRNA targets (22). It is apparent that
the simple presence of an RNABP and a target mRNA in a given cell
is not the sole determinant of their in vivo interaction. The
mechanisms underlying these dynamics are not well understood, but
may include subcellular compartmentalization, posttranslational
modification of components of the RNP, the presence of either
protein or noncoding RNA accessory factors and competition or
cooperation with other posttranscriptional mediators (13, 25).
Previous reports that the RNABP TTP functions interdependently with
miR-16 in ARE-mediated decay of tumor necrosis factor mRNA, and
that HuR can conditionally derepress miR-122 mediated translational
inhibition, also indicate the importance of cellular context in
which to investigate functional interactions between
posttranscriptional mediators (5, 6). The relief of miR-122
repression by HuR resulted in recruitment of the targeted mRNA to
actively translating polysomes, consistent with previous studies
with the neuronal HuB protein (12). Our current results support the
suggestion that miRNA:mRNA interactions are maintained upon HuR
binding of co-targeted transcripts (6). However, it is not known
whether relief of miRNA-mediated translational inhibition is
universally the result of co-targeting of mRNAs by HuR. If HuR
recruitment of these mRNAs to active polysomes is a more general
mechanism of miRNA derepression, and the miRNA interactions with
the mRNAs are maintained, it would provide the potential for
dynamic reversibility of this derepression on a more global level
if cellular conditions change and the HuR association is then lost.
Further studies will be required to understand how HuR and the HuR
RNP-associated miRNA subset reported here influence the contextual
fate of the broader co-associated mRNA populations and resultant
protein expression.
[0070] The data presented here also support a corollary to the
posttranscriptional RNA operon theory (7, 24). A central assertion
of this model is that functionally related genes are co-regulated
combinatorially at the posttranscriptional level by trans-acting
factors such as RNABPs and miRNAs that recognize related regulatory
sequence elements in the respective mRNAs. Our demonstration of the
association of a discrete miRNA subset with a specific group of
target-enriched mRNAs in the HuR mRNP supports this model.
Furthermore, it suggests that RNABP-associated mRNAs may be further
divided into subpopulations based upon potential regulation by
other posttranscriptional mediators such as miRNAs. The added
layers of combinatorial regulation are potentially vast, and may
allow for extensive fine-tuning of gene expression as well as
agility, while maintaining broader canalization of developmental
programs (26).
[0071] HuR is the first RNABP reported to associate with a discrete
subset of miRNAs, in addition to a subset of mRNAs enriched for
predicted targets of miRNAs. As HuR is an established ARE binding
and regulatory protein, these data are consistent with
bioinformatics approaches that have been used to predict the
preferential targeting by human miRNAs of mRNAs containing AU-rich
3'UTRs (27). Moreover, UTR evolution and the robustness of gene
expression programs appear to have been significantly influenced by
posttranscriptional regulators (7-9, 28-30). Our data suggest that
the combinatorial effects of different classes of
posttranscriptional factors may in fact mediate this evolutionary
progression. Given that we find a subpopulation of HuR-associated
mRNAs highly enriched for predicted miRNA targets in mammalian
cells, we propose that many posttranscriptionally regulated mRNAs
may have evolved or acquired sequence elements that enabled
combinatorial regulation via multiple mechanisms. A more thorough
understanding of the coordination of these RNA-RNA and RNA-protein
interactions will require the elucidation of biologically defined
networks involving RNABPs, miRNAs and the messenger RNAs they
co-target.
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TABLE-US-00002 [0101] TABLE 1A HuR associates with seven miRNA
families* Human miRNA Family Seed + m8 HuR-associated miRNAs
miR-181 ACAUUCA miR-181a, miR-181b, miR-181c miR-103 GCAGCAU
miR-103, miR-107 miR-29b AGCACCA miR-29c miR-20 AAAGUGC miR-17-5p,
miR-106a miR-19 GUGCAAA miR-19b miR-15 AGCAGCA miR-16 let-7 GAGGUAG
let-7a, let-7c, let-7d, let-7f *The 14 miRNAs associated with HuR
in Jurkat cells represent 7 families based on seed sequence
conservation. These 7 human miRNA families, the related seed
sequence plus 1 nucleotide (utilized for predicting mRNA targets of
miRNAs), and the HuR-associated miRNAs which are grouped into each
microRNA family are shown.
TABLE-US-00003 TABLE 2 HuR associates with mRNAs enriched for
predicted targets of miRNAs.* Mapped mRNA Targets of HuR- RNA
Source mRNAs associated miRNAs Total RNA 7543 439 HuR mRNP 1013 108
HuR mRNP as % of Total RNA 11% 25%.sup.# *The 7 families of miRNAs
associated with HuR are predicted by the TargetScanS algorithm to
target a subpopulation of mRNAs that are significantly enriched in
the HuR RNP when compared to total cellular RNA. Mapped mRNA
numbers represent those unique mRNAs in total cellular RNA and the
HuR mRNP that were found to overlap between the array platform and
the gene list utilized for TargetScanS predictions. This overlap
consisted of 7543 mRNAs in total RNA and 1013 mRNAs in the HuR
mRNP. P value for enrichment of predicted targets in the HuR mRNP
(2.6e-16) was calculated using a hypergeometric statistical
analysis (see Materials and Methods). .sup.#p value = 2.6e-16
(miRNA target enrichment in HuR mRNP)
TABLE-US-00004 TABLE 3 Predicted mRNA targets of HuR-associated
miRNAs are enriched for specific gene ontology functional
categories.* A. miRNA targets in HuR mRNP B. miRNA targets in total
RNA only GO Category p value GO Category p value Nucleoside,
Nucleotide and 7.30e-06 Nucleoside, Nucleotide and 1.47e-08 Nucleic
Acid Metabolism Nucleic Acid Metabolism Nucleic Acid Binding
7.65e-04 mRNA Transcription 1.36e-07 Other Ligase 6.50e-03 mRNA
Transcription Regulation 1.19e-06 Other Transcription Factor
1.04e-02 Protein Kinase 3.06e-05 Ligase 1.06e-02 Protein
Phosphorylation 4.44e-05 Other Protein Metabolism 1.69e-02
Transcription Factor 5.48e-05 Pre-mRNA Processing 1.94e-02 Protein
Modification 2.49e-04 mRNA Splicing 3.10e-02 Cell Cycle 2.57e-04
mRNA Transcription 3.12e-02 Developmental Process 3.25e-04 Other
Miscellaneous Protein 4.80e-02 Kinase 3.57e-04 Function Protein
Metabolism & 2.42e-03 Modification Non-receptor Serine/
5.66e-03 Threonine Protein Kinase Transcription Cofactor 1.75e-02
General Vesicle Transport 1.77e-02 Intracellular Protein Traffic
1.98e-02 Other Transcription Factor 4.32e-02 *Gene ontology
functional category enrichment for predicted mRNA targets of
HuR-associated miRNAs in (A) HuR mRNP and (B) total RNA only. Gene
list comparisons were carried out using the "PANTHER" database.
Enrichment p values were calculated against NCBI Homo sapiens gene
list using the Binomial statistic with Bonferroni correction for
multiple testing.
Materials and Methods
[0102] Cell Culture and Preparation of Lysates.
[0103] Human acute T cell leukemia Jurkat cells were cultured in
RPMI 1640 supplemented with 10% FBS (GIBCO/BRL). Lysates were
prepared essentially as described (1). Exceptions include the
addition of 10% glycerol to the polysome lysis buffer and passage
of cell lysate through a 27 gauge needle 10 times after
resuspension of harvested cells in lysis buffer.
[0104] IP Assays and Isolation of RNA.
[0105] IP of endogenous HuR and PABP mRNP complexes were used to
assess association of endogenous target mRNAs. Assays were
performed essentially as described (1, 2). IPs utilized 200 .mu.l
pre-swollen and packed Protein-A Sepharose beads (Sigma) loaded
with 60 .mu.g of anti-HuR (3A2) (3), anti-PABP (4), IgG1 (BD
PharMingen) or normal rabbit sera immunoglobulin. Antibody loaded
beads were incubated with 5 mg (total protein) cell lysate for four
hours at 4.degree. C., washed 4 times with ice-cold NT2 buffer (50
mM Tris pH 7.4/150 mM NaCl/1 mM MgCl2/0.05% Nonidet P-40) followed
by 3 washes with ice-cold NT2 supplemented with 1M Urea. Extraction
of associated RNA was performed as described (1), and total RNA was
isolated using the Trizol reagent (GIBCO/BRL). All RNA samples were
divided into two aliquots for subsequent analysis on mRNA or miRNA
arrays.
[0106] mRNA Array Analysis.
[0107] Total and RNP-associated RNA (and negative control IPs) were
assayed for mRNAs on two color Operon Human Oligo Arrays (version
2.1) as described (5). Probe production used direct labeling of
experimental samples (Cy 3) and Stratagene Universal Human
Reference RNA (Cy 5). Results were analyzed using GeneSpring GX 7.3
(Agilent) with per spot and per chip (lowess) normalization. mRNAs
were determined to be components of total RNA or specific
endogenous targets of a given RNABP if present on 2 of 3 biological
replicate arrays at a level of 2 fold above local background in the
experimental channel as well as 10 fold above signal/noise ratio of
parallel negative control IP (IgG1 or normal rabbit sera).
[0108] microRNA Array Analysis.
[0109] Total and RNP-associated RNA (and negative control IPs) were
assayed for miRNAs using a custom array platform capable of
detecting 156 human miRNAs essentially as described (6). Exceptions
include using 10 .mu.g/ml BSA in the labeling buffer and a
reference oligonucleotide concentration of 0.05 .mu.M for labeling.
Arrays were washed once in 2.times.SSC/0.025% SDS at 25.degree. C.,
three times in 0.8.times.SSC at 23.degree. C., and twice in
0.4.times.SSC at 4.degree. C. Computational analysis on each array
was performed as described (6). miRNAs were determined to be
components of total RNA or associated with a given RNABP if present
on 2 of 3 biological replicate arrays at a level of 2 fold above
local background in duplicate spots as well as 10 fold above
signal/noise ratio of parallel negative control IP (IgG1 or normal
rabbit sera).
[0110] microRNA Target Enrichment Analysis.
[0111] miRNA target predictions were taken from the supplementary
data of Lewis et al. (7). This algorithm uses multiple alignments
to identify conserved Watson/Crick hexamer matches to bases 2-7 of
a miRNA, flanked by either a Watson/Crick match to position 8 of
the miRNA or a conserved adenosine in position 1 of the target. We
used the 12928 predictions conserved in 5-species alignments
(human, mouse, rat, dog, and chicken). Lewis et al. provide these
predictions as IDs of cDNAs obtained from the UCSC genome
annotations which may lead to duplicate entries in the form of
several cDNAs reported for one gene. To remove these duplicates, we
mapped the predicted mRNA targets to unique Ensembl gene IDs as of
August 2005, leaving 10182 predictions. Ensembl IDs also allowed us
to match predicted target genes to the mRNA microarray probes
(Table 2). We mapped targets for all 62 miRNA families to 2518
genes represented on the mRNA array platform, 1003 of which were
detected as expressed in Jurkat cells. Enrichment of targeted mRNAs
associated with HuR was determined by hypergeometric tests in
comparison with total cellular RNA.
[0112] Gene Ontology Enrichment Analysis.
[0113] Gene lists of mRNAs predicted to be targets of
HuR-associated miRNAs were compared against the complete NCBI H.
sapiens gene list using the Panther database (8). Significant
enrichment in a functional category was determined using the
Binomial statistic with Bonferroni correction for multiple testing
(p value <0.05).
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[0122] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
Sequence CWU 1
1
717RNAArtificialMicroRNA 1acauuca 7 27RNAArtificialMicroRNA
2gcagcau 7 37RNAArtificialMicroRNA 3agcacca 7
47RNAArtificialMicroRNA 4aaagugc 7 57RNAArtificialMicroRNA 5gugcaaa
7 67RNAArtificialMicroRNA 6agcagca 7 77RNAArtificialMicroRNA
7gagguag 7
* * * * *